Methods and compositions for monitoring phagocytic activity

ABSTRACT

The invention relates to compositions and methods for monitoring phagocytic activity (e.g., diseases and conditions relating to phagocytic activity). In particular, the invention relates to compositions and methods for diagnosing, monitoring, and/or assessing risk of neurodegenerative diseases (e.g., AD).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/989,950, filed Jun. 10, 2013, now allowed as U.S. Pat. No.11,231,426, which is a 371 U.S. National Entry of International PatentApplication No. PCT/IB2011/002999, filed Nov. 28, 2011. which claimspriority to U.S. Provisional Patent Application No. 61/417,559, filedNov. 29, 2010, the contents of which are hereby incorporated byreference in their entireties.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 2,000 Byte ASCII (Text) file named“31699-303_ST25” created on Jan. 24, 2022.

FIELD OF THE INVENTION

The invention relates to compositions and methods for monitoringphagocytic activity (e.g., diseases and conditions relating tophagocytic activity). In particular, the invention relates tocompositions and methods for diagnosing, monitoring, and/or assessingrisk of neurodegenerative diseases (e.g., Alzheimer's disease (AD)).

BACKGROUND OF THE INVENTION

AD, the most common cause of dementia, is an acquired cognitive andbehavioral impairment that interferes with social and occupationalfunctioning. AD is a major public health problem from the economicperspective, causing not only tremendous economic burden relating topatients themselves, but also due to social, economic, physical, andpsychological impacts on caregivers. In the United States, the cost ofcaring for patients with dementia was $144 billion per year in 2009, andthe average yearly cost for healthcare and long-term care services perpatient was about $33,007 in 2004, the most recent data available(Alzheimer's Association (2010) Alzheimer's & Dementia 6:158-194).

AD affects approximately 5.3 million people in the United States as of2010 (Alzheimer's Association (2010) Alzheimer's & Dementia 6:158-194)and 26.6 million individuals worldwide as of 2006. A larger number ofindividuals have decreased levels of cognitive function (e.g.,measurable mild cognitive impairment (MCI) or even earlier onlysubjective cognitive impairment (SCI)) that frequently evolve into afull-blown dementia, thereby increasing the number of affected persons.By 2030, an estimated 7.7 million Americans aged 65 and older will haveAD (Hebert et al. (2003) Arch. Neurol. 60(8):1119-22). Statisticalprojections indicate that the number of persons affected by the disorderin the United States could range from 11-16 million by the year 2050(Hebert et al. (2003) Arch. Neurol. 60(8):1119-22).

The causative factors of sporadic AD remain elusive, but diseaseprogression is correlated with hallmark anatomic pathological signsincluding neurofibrillary tangles (NFTs); senile plaques (SPs) at themicroscopic level; and cerebrocortical atrophy. Amyloid deposition isalso implicated in two other common causes of dementia, vasculardementia (VaD) and dementia with Lewy bodies (DLB), as well as inclusionbody myositis, a muscle disease. Although AD follows a typical course ofprogression, there is a dearth of definitive diagnostic methods,particularly for early-stage AD. This is particularly problematic sinceMCI and early-stage AD are clinically valuable periods forimplementation of disease-modifying therapies (Vellas et al. (2007)Lancet Neurol. 6:56-62). This is significant because the widespreaddamage caused by AD is most likely irreversible at later stages ofdisease progression (Braak et al. (1991) ActaNeuropathol. 82:239-259).

Improved methods and compositions are needed for detection, diagnosis,and monitoring of neurodegenerative diseases, e.g., AD.

SUMMARY OF THE INVENTION

The invention relates to compositions and methods for monitoringphagocytic activity (e.g., diseases and conditions relating tophagocytic activity). In particular, the invention relates tocompositions and methods for detecting, diagnosing, monitoring, and/orassessing risk of neurodegenerative diseases (e.g., AD).

Numerous diseases and conditions involve activation of macrophages andproteolysis of proteins, e.g., pathologic aggregates of endogenousproteins. For example, in AD, the formation of amyloid plaques in thebrain occurs when aggregates of amyloid beta (Aβ) peptides are formedfollowing proteolysis of amyloid precursor protein (APP).

Immunization of an individual with Aβ peptides triggers phagocytosis(Schenk et al. (1999) Nature 400:173-177; herein incorporated byreference in its entirety). Immune activity may also be involved in thenatural evolution of amyloid pathology, e.g., the continuous level ofphagocytic activity may contribute to plaque load over time. There isevidence that macrophages circulate from the bone marrow to the centralnervous system in pathological conditions and contribute to plaqueclearing in mouse models of AD (Simard et al. (2006) Neuron 49:489-502;herein incorporated by reference in its entirety), and macrophagesisolated from AD patients exhibit reduced phagocytosis of Aβ peptidescompared to macrophages from non-AD patients (Fiala et al. (2005) J.Alzheimers Dis. 7:221-232; herein incorporated by reference in itsentirety). Experiments conducted during the course of developing someembodiments of the present invention identified Aβ peptides present inactivated macrophages isolated from peripheral blood samples (e.g., SEQID NOs. 1-5). The peptides range from 6 to 11 amino acids in length.Accordingly, in some embodiments, determination of the level, presence,or absence of Aβ peptides (e.g., SEQ ID NOs.:1-5) in leukocytes ormacrophages (e.g., activated macrophages) of a patient finds use asmethod for diagnosing, monitoring, and/or assessing the risk of disease(e.g., AD, vascular dementia, dementia with Lewy bodies, inclusion bodymyositis).

In some embodiments, methods of the present invention comprise obtaininga sample from a subject (e.g., a human subject), without limitation tothe sample type. In preferred embodiments, the sample comprisesleucocytes and/or macrophages (e.g., activated macrophages). The samplemay comprise blood (e.g., peripheral blood), cerebrospinal fluid (CSF),tissue (e.g., lung, spleen, liver, brain, pancreas, small intestine,tumour). In some embodiments, the sample is a bronchial alveolar lavage(BAL) sample. In some embodiments, the sample is bone marrow. Inparticularly preferred embodiments, the sample is a blood sample or aCSF sample.

In some embodiments, methods of the present invention comprise a step ofisolating or enriching for macrophages (e.g., activated macrophages),without limitation to the technique used for such isolation orenrichment (e.g., as described in Macrophages (2000) ed. Paulnock, D.M., Oxford Univ. Press, Oxford, UK). Examples of macrophage isolation orpurification techniques include but are not limited to adhesion oradherence methods (e.g., adherence to solid support (e.g., plastic,glass) whether such support is coated (e.g., coated with gelatine,microexudate, collagen, lysine) or uncoated), differentialcentrifugation (e.g., gradient centrifugation, isopycnic gradientcentrifugation, Ficoll-Hypaque gradient centrifugation, Percoll gradientcentrifugation), flow cytometry, fluorescent activated cell sorting(FACS), flotation techniques, antibody-mediated affinity techniques(e.g., immunoprecipitation, antibody-mediated binding to solid support(e.g., magnetic beads, non-magnetic particles, plates, wells, cards,chips, slides, arrays, etc.). In some embodiments, the macrophage (e.g.,activated macrophage) purification or isolation technique involvesbinding of macrophages with a binding partner (e.g., antibody) that isnon-covalently or covalently associated with a moiety used for direct orindirect detection (e.g., a fluorescent tag, biotin, streptavidin, aradioisotope, an epitope, an affinity tag).

In some embodiments, methods of the present invention comprise detectionof the level of one or more biomarkers of a condition or disease state(e.g., AD) present in leucocytes and/or macrophages (e.g., activatedmacrophages), without limitation to the technique used for suchdetection. In preferred embodiments, one or more of the biomarkers areAβ peptides (e.g., SEQ ID.NOs.:1-5). In some embodiments, the level ofone or more biomarkers (e.g., Aβ peptides, SEQ ID.NOs.:1-5) isdetermined quantitatively. In some embodiments, the presence or absenceof one or more biomarkers is determined qualitatively. In someembodiments, a decrease in the level(s) of one or more biomarkers (e.g.,in a sample of tissue or biological fluid, in a sample enriched forleucocytes and/or macrophages (e.g., activated macrophages)) relative toa control is correlated with an increased risk of disease (e.g., AD),diagnosis of disease (e.g., AD). In some embodiments, samples comprisingmacrophages and/or microglia are assessed for level of phagocyticactivity, e.g., are exposed to a first polypeptide (e.g., an Aβ peptide,APP) for a period of time and the level of phagocytosis of thepolypeptide is determined. In some embodiments, the presence, absence,or level of polypeptides smaller in length than the first polypeptide(e.g., a second Aβ peptide, SEQ ID NOs:1-5) serves as an indication ofthe level of phagocytic and/or proteolytic activity in the originalsample.

In some embodiments, methods of the present invention involve comparingthe level of one or more biomarkers (e.g., Aβ peptides, SEQ ID.NOs.:1-5)in samples from the same patient at one or more different time points(e.g., longitudinal sampling), e.g., where such levels are correlatedwith disease progression. In some embodiments, methods of the presentinvention involve comparing the level of one or more biomarkers (e.g.,Aβ peptides, SEQ ID.NOs.:1-5) in a sample from a patient relative to thelevel(s) found in a sample from a control subject lacking disease (e.g.,AD). In some embodiments, methods of the present invention involvecomparing the level of one or more biomarkers (e.g., Aβ peptides, SEQID.NOs.:1-5) in a sample from a first subject relative to the average,mean, or other statistically determined level(s) (e.g., “standardlevel(s)”) found in samples from a plurality of control subject slackingdisease (e.g., AD), wherein the level of the one or more biomarker(s) inthe first subject is abnormal if there is a statistically significantdifference from the standard level.

In some embodiments, the prevent invention relates to compositionscomprising isolated Aβ peptides with greater than 80%, greater than 85%,greater than 90%, preferably greater than 95%, most preferably greaterthan 99% identity to SEQ ID.NOs.:1-5, and agent(s) used for theirspecific detection (e.g., antibodies capable of specific detection of Aβpeptides with greater than 80%, greater than 85%, greater than 90%,preferably greater than 95%, most preferably greater than 99% identityto SEQ ID.NOs.:1-5). In some embodiments, the present invention relatesto kits comprising isolated Aβ peptides with greater than 80%, greaterthan 85%, greater than 90%, preferably greater than 95%, most preferablygreater than 99% identity to SEQ ID.NOs.:1-5 and/or agent(s) used fortheir specific detection (e.g., antibodies capable of specific detectionof Aβ peptides with greater than 80%, greater than 85%, greater than90%, preferably greater than 95%, most preferably greater than 99%identity to SEQ ID.NOs.:1-5).

In certain embodiments, the present invention provides a compositioncomprising an isolated peptide, the isolated peptide being 6 to 11 aminoacids in length, the peptide having at least 80% identity to an aminoacid sequence such as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, or SEQ ID NO:5. In some embodiments, the peptide has at least 85%identity to an amino acid sequence such as SEQ ID NO:1, SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, or SEQ ID NO:5. In some embodiments, the peptidehas at least 90% identity to an amino acid sequence such as SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5. In someembodiments, the peptide has at least 95% identity to an amino acidsequence such as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, orSEQ ID NO:5. In some embodiments, the peptide has at least 99% identityto an amino acid sequence such as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:4, or SEQ ID NO:5.

In certain embodiments, the present invention provides a kit for thedetection, diagnosis, and/or monitoring of a disease, the kit comprisingcomponents such as one or more synthetic peptides having at least 80%identity to an amino acid sequence such as SEQ ID NO:1, SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, or SEQ ID NO:5 and one or more first bindingreagents specifically capable of binding to a peptide having at least80% identity to an amino acid sequence such as SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4 or and SEQ ID NO:5. In some embodiments, thedisease is a type such as AD, vascular dementia, or dementia with Lewybodies. In some embodiments, the binding reagent is an antibody. In someembodiments, the kit further comprises components such as syntheticAβ₄₂, media, a macrophage-specific binding reagent, or a secondarybinding reagent capable of the one or more first binding reagents.

In certain embodiments, the present invention provides a method fordiagnosing, monitoring, and/or assessing the risk of a neurodegenerativedisease in a subject, the method comprising 1) obtaining a sample from asubject, the sample comprising a cell type such as leucocytes,monocytes, macrophages, or activated macrophages, and b) detecting thelevel of a peptide in the sample, wherein the peptide has at least 80%identity to an amino acid sequence such as SEQ ID NO:1, SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, or SEQ ID NO:5, wherein an abnormal level of thepeptide relative to a standard level is indicative of the presence orseverity of the neurodegenerative disease in the subject. In someembodiments, the neurodegenerative disease is a type such as AD,vascular dementia, and dementia with Lewy bodies. In some embodiments,the sample is a type such as a blood sample, a tissue sample, or acerebrospinal fluid sample. In some embodiments, the cell type ismacrophage. In some embodiments, the cell type is activated macrophage.In some embodiments, the standard level a type such as the level in thesubject at a different time point, the level in a different subjectlacking the neurodegenerative disease, and the average level in aplurality of subjects lacking the neurodegenerative disease. In someembodiments the method further comprises isolating the cell type suchasleucocytes, monocytes, macrophages, or activated macrophages. In someembodiments, the isolated cells are macrophages. In some embodiments,the method further comprises incubating the isolated cells withsynthetic Aβpolypeptide. In some embodiments, the syntheticAβpolypeptide is Aβ₄₂.

Additional embodiments will be apparent to persons skilled in therelevant art based on the teachings contained herein.

DESCRIPTION OF THE DRAWINGS

FIG. 1 provides adepiction of Aβ content in monocytes.

FIG. 2 provides data associating intracellular content of Aβ withclinical diagnosis.

FIG. 3 provides a dose response curve typical for an in vitrophagocytosis assay shows confocal microscopy of a macrophage incubatedwith 0.5 ng/μL Aβ₁₋₄₂ overnight and labelled with green fluorescingAβ₁₋₄₂-specific antibodies (6E10 and 4G8). Left, fluorescent signalobserved during immunoconfocal microscopy. Right, transillumination.

FIG. 4 shows a representation of the Aβ-peptides identified through massspectrometry (MS)-analysis as described herein. The central shadedfields show the location of the individual peptides within theAβ₁₋₄₂-molecule. The overlapping shaded region to the right of thecentral shaded region shows the transmembrane region. All peptidesborder the intramembrane (transmembrane) region of the molecule.

FIG. 5 shows results of mass spectrometry (MS)-analysis duringexperiments conducted during the course of developing some embodimentsof the present invention.

The upper panel shows the crude MS-spectrum from lysosomes of cellsincubated in the presence of Aβ₁₋₄₂ and from control cells. The lowerpanel shows further analysis of MS-data from the upper panel based onthe Aβ₁₋₄₂ sequence; grey bars indicate potential fragments and darkshaded squares indicate significant hits within these fragments.

FIG. 6 provides a schematic depiction of an assay for detection andquantification of Alzheimer specific APP metabolites using real timePCR.

FIG. 7 provides an amplification plot from real-time PCR

FIG. 8A and FIG. 8B provide histograms for THP-1 cells afterflowcytometry. Histogram 8 a shows THP-1 cells added Aβ₁₋₄₂ in medium.Histogram 8 b shows THP-1 cells without Aβ₁₋₄₂ in medium. Theintracellular Aβ content is illustrated in the histograms. Isotypemedian Abeta FITC-A signal is 102 (Results not shown). A shift in AbetaFITC-A to the right compared to the isotype is indicating cells withpresence of intracellular Aβ. The ellipse in histogram 8 a showspresence of a tail of Aβ positive cells that is absent in histogram 8 b.This tail is indicating a higher amount of cells containing phagocytosedintracellular Aβ in histogram 8 b.

FIG. 9 provides a standard PCR curve showing threshold cycle (Ct value)vs. log peptide concentration. The graph shows the threshold cycle (Ctvalue) against log concentration. Equation for the curve isy=−0.6056x+32,629. R²=0.8653.

FIG. 10A and FIG. 10B provide histograms for monocytes/macrophages frompatient and control after flowcytometry. 10 a shows patient samplestained intracellular with 6E10/4G8. 10 b shows control sample stainedintracellular with 6E10/4G8. The intracellular Aβ₁₋₄₂ content inmacrophages/monocytes from patient and control is illustrated in thehistograms. Isotype median Abeta FITC-A signal is 102 (Results notshown). A shift in Abeta FITC-A to the right compared to the isotype isindicating cells with presence of intracellular Aβ₁₋₄₂. Histograms 3 and4 are indicating presence of approx. of intracellular Aβ₁₋₄₂ whencompared to the isotypes.

FIG. 11 provides a standard PCR curve showing threshold cycle (Ct value)vs. log peptide concentration. The graph shows the threshold cycle (Ctvalue) against log concentration. Equation for the curve isy=−3,8956x+37,275. R²=08818.

Definitions

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

As used herein, the term “sensitivity” is defined as a statisticalmeasure of performance of an assay (e.g., method, test), calculated bydividing the number of true positives by the sum of the true positivesand the false negatives.

As used herein, the term “specificity” is defined as a statisticalmeasure of performance of an assay (e.g., method, test), calculated bydividing the number of true negatives by the sum of true negatives andfalse positives.

As used herein, the term “informative” or “informativeness” refers to aquality of a marker or panel of markers, and specifically to thelikelihood of finding a marker (or panel of markers) in a positivesample.

As used herein, “an individual is suspected of being susceptible at riskfor AD” is meant to refer to an individual who is at an above-averagerisk of developing AD (AD). Examples of individuals at a particular riskof developing AD are those whose family medical history indicates aboveaverage incidence of AD, individuals of advanced age, individualsexhibiting signs or symptoms of MCI or SCI. Other factors which maycontribute to an above-average risk of developing AD may be based uponan individual's specific genetic, medical, psychological, psychosocial,and/or behavioral background and characteristics.

As used herein, the term “isolated” when used in relation to material(e.g., a cell, a leukocyte, a macrophage, an activated macrophage, amicroglia) refers to a material that is identified and separated from atleast one component or contaminant with which it is ordinarilyassociated in its natural source. An isolated material is such presentin a form or setting that is different from that in which it is found innature.

As used herein, the terms “immunoglobulin” and “antibody” refer toproteins that bind a specific antigen. Immunoglobulins include, but arenot limited to, polyclonal, monoclonal, chimeric, and humanizedantibodies, Fab fragments, F(ab′)₂ fragments, and includesimmunoglobulins of the following classes: IgG, IgA, IgM, IgD, IbE, andsecreted immunoglobulins (sIg). Immunoglobulins generally comprise twoidentical heavy chains and two light chains. However, the terms“antibody” and “immunoglobulin” also encompass single chain antibodiesand two chain antibodies.

As used herein, the term “antigen binding protein” refers to proteinsthat bind to a specific antigen. “Antigen binding proteins” include, butare not limited to, immunoglobulins, including polyclonal, monoclonal,chimeric, and humanized antibodies; Fab fragments, F(ab′)₂ fragments,and Fab expression libraries; and single chain antibodies.

The term “epitope” as used herein refers to that portion of an antigenthat makes contact with a particular immunoglobulin.

The terms “specific binding” or “specifically binding” when used inreference to the interaction of an antibody and a protein or peptidemeans that the interaction is dependent upon the presence of aparticular structure (i.e., the antigenic determinant or epitope) on theprotein; in other words the antibody is recognizing and binding to aspecific protein structure rather than to proteins in general. Forexample, if an antibody is specific for epitope “A,” the presence of aprotein containing epitope A (or free, unlabelled A) in a reactioncontaining labeled “A” and the antibody will reduce the amount oflabeled A bound to the antibody.

As used herein, the terms “non-specific binding” and “backgroundbinding” when used in reference to the interaction of an antibody and aprotein or peptide refer to an interaction that is not dependent on thepresence of a particular structure (i.e., the antibody is binding toproteins in general rather that a particular structure such as anepitope).

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to, humans, non-human primates,rodents, and the like (e.g., that is to be the recipient of a particulartreatment (e.g., transplant graft) or that is a donor of a graft. Theterms “subject” and “patient” are used interchangeably in reference to ahuman subject, unless indicated otherwise herein (e.g., wherein asubject is a graft donor).

As used herein, the term “sample” is used in its broadest sense. Forexample, in some embodiments, it is meant to include a specimen (e.g.,blood sample, cerebrospinal fluid (CSF) sample). In preferredembodiments, it is meant to include a biological sample.

The present invention is not limited by the type of biological sampleused or analyzed. The present invention is useful with a variety ofbiological samples including, but are not limited to, tissue (e.g.,organ (e.g., heart, liver, brain, lung, stomach, intestine, spleen,kidney, pancreas, and reproductive (e.g., ovaries) organs; lung biopsy),glandular, skin, and muscle tissue), cell (e.g., blood cell (e.g.,lymphocyte or erythrocyte), muscle cell, tumor cell, bronchial cell,bronchioalveolar cells, and skin cell), gas, bodily fluid (e.g.,tracheal aspirate fluid, bronchoalveolar fluid, bronchoalveolar lavagesample, blood or portion thereof, serum, plasma, urine, semen, saliva,etc), or solid (e.g., stool) samples obtained from a human (e.g., adult,infant, or embryo) or animal (e.g., cattle, poultry, mouse, rat, dog,pig, cat, horse, and the like). Biological samples may be obtained fromall of the various families of domestic animals, as well as feral orwild animals, including, but not limited to, such animals as ungulates,bear, fish, lagamorphs, rodents, etc.

Biological samples also include biopsies and tissue sections (e.g.,biopsy or section of tumor, growth, rash, infection, orparaffin-embedded sections), medical or hospital samples (e.g.,including, but not limited to, bronchoalveolar lavage fluid (BAL)samples, tracheal aspirate fluid, blood samples, saliva, buccal swab,cerebrospinal fluid, pleural fluid, milk, colostrum, lymph, sputum,vomitus, bile, semen, oocytes, cervical cells, amniotic fluid, urine,stool, hair and sweat), and laboratory samples (e.g., subcellularfractions).

As used herein, the term “purified” or “to purify” refers to the removalof components (e.g., contaminants) from a sample. For example,antibodies are purified by removal of contaminating non-immunoglobulinproteins; they are also purified by the removal of immunoglobulin thatdoes not bind to the target molecule. The removal of non-immunoglobulinproteins and/or the removal of immunoglobulins that do not bind to thetarget molecule results in an increase in the percent of target-reactiveimmunoglobulins in the sample. In another example, recombinantpolypeptides are expressed in bacterial host cells and the polypeptidesare purified by the removal of host cell proteins; the percent ofrecombinant polypeptides is thereby increased in the sample. In yet afurther example, a specific cell type (e.g., leukocyte, macrophage,activated macrophage, microglial cell) may be purified or enriched forusing a cell sorting technique (e.g., flow cytometery,fluorescence-activated cell sorting (FACS)).

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity or more (e.g., 99percent sequence identity). Preferably, residue positions that are notidentical differ by conservative amino acid substitutions. Conservativeamino acid substitutions refer to the interchangeability of residueshaving similar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions, methods and kits relatingto the detection, monitoring, diagnosis, or assessment of risk ofdeveloping a neurodegenerative disease (e.g., AD, vascular dementia,dementia with Lewy bodies, inclusion body myositis, or otherneurodegenerative disease). In some embodiments, the present inventionfinds use in characterizing alterations in phagocytic activity ofmacrophages (e.g., phagocytosis of pathological polypeptides, e.g.,phagocytosis of Aβ-peptides, phagocytosis of amyloid precursor protein).In some embodiments, the invention comprises methods for diagnosingmonocyte/macrophage phagocytic activity in samples from body fluids,e.g., by detection of the level, presence, or absence of phagocytosedpeptide fragments (e.g., Aβ-peptides, SEQ ID NOs:1-5).

In experiments conducted during the course of developing someembodiments of the present invention, it was shown that blood-derivedmonocytes/macrophages internalizeAβ₁₋₄₂ in an in vitro setting. Adepiction of Aβ content is provided in FIG. 1. FIG. 2 provides dataassociating intracellular content of Aβ with clinical diagnosis. FIG. 3provides a dose response curve typical for an in vitro phagocytosisassay and shows that the internalization occurs via theendosomal-lysosomal system as the Aβ₁₋₄₂-spesific green fluorescenceappears in intracellular granula; see also (Boland et al.(2010), inpress, doi: 10.1074/jbc.M110.186411;Lorenzen et al. (2010) Mol. Brain3:11; each herein incorporated by reference in its entirety). It ispostulated that method of evaluating the efficiency of said clearance isneeded to improve the diagnostic process, e.g., when diagnosing AD.

In some embodiments, the present invention provides methods fordiagnosing monocyte/macrophage phagocytic activity in samples, e.g. bodyfluid samples, by detection of phagocytosed peptide-fragments.Experiments conducted during the course of developing some embodimentsof the present invention allowed identification of peptide fragmentstructures specific for phagocytosed and partially degraded amyloidprecursorprotein (APP), e.g., as described in Example 1 (e.g., SEQ IDNOs.:1-5). In some embodiments, the peptides are from about 5 to about9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length,with the proviso that a portion of the peptide is at least 80%, 90%, or100% identical to one of SEQ ID NOs:1-5. In some embodiments, theportion of the peptide that is at least 80%, 90%, or 100% identical toone of SEQ ID NOs:1-5 is flanked by N or C-terminal amino acid segments.In some embodiments, Aβ peptide fragments (e.g., SEQ ID NOs:1-5) aresynthesized (e.g., chemically synthesized, synthesized by recombinantexpression), and animals are immunized with individual peptides (e.g.,peptides encoded by SEQ ID NOs:1-5) in order to produce peptide-specificantibodies. In some embodiments, such peptide-specific antibodies may beused in a kit (e.g., a kit for the diagnosis, monitoring, or determiningthe risk of developing) for a disease (e.g., AD) as described in moredetail below.

Accordingly, in some embodiments, the present invention providesimmunoglobulins that bind to the peptides that are at least 80%, 90%, or100% identical to the peptides encoded by SEQ ID NOs:1-5. In someembodiments, the peptides are from 5 to 20 amino acids in length and thepeptides that are at least 80%, 90%, or 100% identical to the peptidesencoded by SEQ ID NOs:1-5 provide at least a portion of an epitopewithin the larger peptide sequence. As used herein, an “antibody orpolyclonal antibody” means a protein that is produced in response toimmunization with an antigen or receptor. The term “monoclonal antibody”means an immunoglobulin derived from a single clone of cells. Allmonoclonal antibodies derived from the clone are chemically andstructurally identical, and specific for a single antigenic determinant.The hybridoma cell lines producing the monoclonal antibodies also arewithin the scope of this invention.

Laboratory methods for producing polyclonal antibodies and monoclonalantibodies, as well as deducing their corresponding nucleic acidsequences, are known in the art, see Harlow and Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, New York (1988) andSambrook et al. (1989) supra. The monoclonal antibodies of thisinvention can be biologically produced by introducing a peptide asdescribed above or a fusion protein or larger protein fragmentcomprising the peptide into an animal, e.g., a mouse or a rabbit. Theantibody producing cells in the animal are isolated and fused withmyeloma cells or heteromyeloma cells to produce hybrid cells orhybridomas. Accordingly, the hybridoma cells producing the monoclonalantibodies of this invention also are provided.

Thus, using the peptides described above, and well known methods, one ofskill in the art can produce and screen the hybridoma cells andantibodies of this invention for antibodies having the ability to bindto the desired peptide (e.g., SEQ ID NOs:1-5).

If a monoclonal antibody being tested binds with the peptide, then theantibody being tested and the antibodies provided by the hybridomas ofthis invention are equivalent. It also is possible to determine withoutundue experimentation, whether an antibody has the same specificity asthe monoclonal antibody of this invention by determining whether theantibody being tested prevents a monoclonal antibody of this inventionfrom binding the peptide with which the monoclonal antibody is normallyreactive. If the antibody being tested competes with the monoclonalantibody of the invention as shown by a decrease in binding by themonoclonal antibody of this invention, then it is likely that the twoantibodies bind to the same or a closely related epitope. Alternatively,one can pre-incubate the monoclonal antibody of this invention with thepeptide with which it is normally reactive, and determine if themonoclonal antibody being tested is inhibited in its ability to bind theantigen. If the monoclonal antibody being tested is inhibited then, inall likelihood, it has the same, or a closely related, epitopicspecificity as the monoclonal antibody of this invention.

The term antibody also is intended to include antibodies of a differentisotype than the monoclonal antibody of this invention. Particularisotypes of a monoclonal antibody can be prepared either directly byselecting from the initial fusion, or prepared secondarily, from aparental hybridoma secreting a monoclonal antibody of different isotypeby using the sib selection technique to isolate class switch variantsusing the procedure described in Steplewski et al. (1985) Proc. Natl.Acad. Sci. 82:8653 or Spira et al. (1984) J. Immunol. Methods 74:307.Thus, the monoclonal antibodies of this invention would includeclass-switch variants having specificity the desired peptide (e.g., SEQID NOs:1-5).

This invention also provides biological active fragments of thepolyclonal and monoclonal antibodies described above. These antibodyfragments retain some ability to selectively bind with its antigen orimmunogen. Such antibody fragments can include, but are not limited to:(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule produced by digestion with the enzymepapain to yield an intact light chain and a portion of one heavy chain;(2) Fab′, the fragment of an antibody molecule obtained by treating withpepsin, followed by reduction, to yield an intact light chain and aportion of the heavy chain; two Fab′ fragments are obtained per antibodymolecule; (3) (Fab′)₂, the fragment of the antibody that is obtained bytreating with the enzyme pepsin without subsequent reduction; F(ab′)₂ isa dimer of two Fab′ fragments held together by two disulfide bonds; (4)Fv, defined as a genetically engineered fragment containing the variableregion of the light chain and the variable region of the heavy chainexpressed as two chains; and (5) SCA, defined as a geneticallyengineered molecule containing the variable region of the light chain,the variable region of the heavy chain, linked by a suitable polypeptidelinker as a genetically fused single chain molecule.

Specific examples of “biologically active antibody fragment” include theCDR regions of the antibodies. Methods of making these fragments areknown in the art, see for example, Harlow and Lane, (1988) supra.

The antibodies of this invention also can be modified to create chimericantibodies (Oi, et al. (1986) BioTechniques 4(3):214). Chimericantibodies are those in which the various domains of the antibodies'heavy and light chains are coded for by DNA from more than one species.

The antibodies of this invention can be linked to a detectable agent ora hapten. The complex is useful to detect the peptides in a sample usingstandard immunochemical techniques such as immunohistochemistry asdescribed by Harlow and Lane (1988) supra and by the methods describesin more detail below. Examples of types of immunoassays which canutilize monoclonal antibodies of the invention are competitive andnon-competitive immunoassays in either a direct or indirect format.Examples of such immunoassays are the enzyme linked immunoassay (ELISA)radioimmunoassay (RIA) and the sandwich (immunometric) and immuno-PCRassays. Detection of the peptide(s) using the monoclonal antibodies ofthe invention can be done utilizing immunoassays which are run in eitherthe forward, reverse, or simultaneous modes, includingimmunohistochemical assays on physiological samples. Those of skill inthe art will know, or can readily discern, other immunoassay formatswithout undue experimentation.

Another technique which may also result in greater sensitivity consistsof coupling the antibodies to low molecular weight haptens. Thesehaptens can then be specifically detected by means of a second reaction.For example, it is common to use such haptens as biotin, which reactsavidin, or dinitropherryl, pyridoxal, and fluorescein, which can reactwith specific anti-hapten antibodies. See Harlow and Lane (1988) supra.

The monoclonal antibodies of the invention can be bound to manydifferent carriers. Examples of well-known carriers include glass,polystyrene, polypropylene, polyethylene, dextran, nylon, amylases,natural and modified celluloses, polyacrylamides, agaroses andmagnetite. The nature of the carrier can be either soluble or insolublefor purposes of the invention. Those skilled in the art will know ofother suitable carriers for binding monoclonal antibodies, or will beable to ascertain such, using routine experimentation.

There are many different labels and methods of labeling known to thoseof ordinary skill in the art. Examples of the types of labels which canbe used in the present invention include enzymes, radioisotopes,fluorescent compounds, colloidal metals, chemiluminescent compounds, andbioluminescent compounds. Those of ordinary skill in the art will knowof other suitable labels for binding to the monoclonal antibody, or willbe able to ascertain such, using routine experimentation. Furthermore,the binding of these labels to the monoclonal antibody of the inventioncan be done using standard techniques common to those of ordinary skillin the art.

In some embodiments, the present invention facilitates the diagnosis,monitoring, and/or determination of risk of developing a disease (e.g.,AD) based upon the level of biomarker peptides (e.g., Aβ peptidefragments (e.g., SEQ ID NOs:1-5) in cells (e.g., macrophages, activatedmacrophages). In some embodiments, the cells (e.g., macrophages,activated macrophages) are isolated from blood (e.g., peripheral blood)and/or cerebrospinal fluid. In some embodiments, the period of timeelapsing between cell (e.g., macrophage, activated macrophage) isolationand testing is less than 5 days, less than 4 days, less than 3 days,less than 2 days, less than 1 day, less than 18 hours, less than 12hours, less than 8 hours, preferably less than 6 hours, and mostpreferably less than 4 hours. In some embodiments, one or moresubcellular fractions are isolated from a cell type (e.g., macrophage,activated macrophage) isolated or purified from a sample. In someembodiments, the subcellular fraction is a lysosomal fraction. In someembodiments, methods of the present invention are combined with exvivoassessment of phagocytosis activity (e.g., as described in Example 1and in PCT App. No. PCT/EP09/001210, herein incorporated by reference inits entirety). In some embodiments, such assessment of phagocytosisactivity comprises incubation of cells (e.g., macrophages, activatedmacrophages) from a sample with or without peptides (e.g., Aβ peptidefragments (e.g., SEQ ID NOs:1-5). In some embodiments, phagocytosis isassessed using techniques such as fluorescence-activated cell sorting(FACS) and/or confocal microscopy. In some embodiments, methods of thepresent invention include the use of techniques such asimmunoprecipitation alone or in combination with mass spectrometry(e.g., IP-MS). In some embodiments, methods of the present inventioncomprise use of antibodies specific to Aβ peptide fragments (e.g., SEQID NOs:1-5). In some embodiments, methods of the present invention finduse in monitoring treatment efficacy and/or disease progress. In someembodiment, methods of the present invention find use in diagnosing,monitoring, and/or assessing the risk of developing diseases involvingmonocyte/macrophage activation, extravasation, phagocytosis and re-entryto the circulation. In some embodiments, methods of the presentinvention find use in monitoring the phagocytosis of amelyoid precursorprotein (APP). While the present invention is not limited to anyparticular mechanism, and an understanding of the mechanism is notnecessary to practice the present invention, it is contemplated that APPphagocytosis is a central mechanism in brain APP-amyloid homeostasisaltered during the progression of Alzheimer' disease.

An embodiment of the present invention will now be described. A sampleof cerebrospinal fluid (CSF) is obtained from a patient by lumbarpuncture. The macrophages in the sample are stained withfluorescent-labeled anti-CD 14 and anti-CD 16 antibodies and themacrophages are then withdrawn from the sample by fluorescence activatedcell sorting.

The cells are selected on the basis of CD 14 and CD 16 expressionbecause this enables activated macrophages to be differentiated fromquiescent cells (increasing CD 16 expression signifies an activatedstatus). This technique also avoids the inadvertent sampling of othercell types such as CD68 positive dendritic cells. This approachcontrasts with that reported by Fiala et al (39) in which CD68 positivecells were selected.

The resultant macrophage cells are lysed and prepared for proteinanalysis. The cell lysate is mixed with monoclonal antibodies capable ofbinding fragments of the Aβ protein (SEQ. ID NO.:6,DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL MVGGVVIA). Exemplary antibodies are6E10, 4G8 and 11A50-B10 from Signet Laboratories, Inc., and theantibodies specific for example, SEQ ID Nos:1-5 described above. The6E10 antibody is used to immunoprecipitate Aβ fragments 1-16, the 4G8antibody immunoprecipitates Aβ fragments 17-24 and the 1 1A50-B10antibody immunoprecipitates Aβ fragments 1-40. In alternativeembodiments, a different panel of antibodies, specific for otherfragments, may be used. The monoclonal antibodies are also coupled tomagnetic beads, for example, with beads bound to anti-IgG antibodies.The magnetic beads are used to extract the fragments of the Aβ protein.The antibodies and beads are subsequently removed from the peptidefragments. The peptide fragments are then analyzed by MALDI-TOF massspectrometry and the sequence of the fragments derived from themolecular mass of each fragment. The results are displayedquantitatively to indicate the relative quantity of each fragment. Whereno Aβ protein or Aβ protein fragments are detected in the macrophages,this is indicative that the patient has AD.

The Aβ fragments shown in the IP-MS spectra result from intracellulardegradation according to the character of the catalytic active site andconditions of action of intracellular protease/peptidases. Suchfragments do not necessarily correspond to the sequences of Aβ fragmentsfound extracellularly in CSF. Thus in some embodiments, in order toidentify the exact length of each Aβ fragment obtained in theexperiment, the peptides are isolated for determination of theirrespective amino acid sequences.

It is to be appreciated that the method described above detects thepresence of the Aβ protein fragments that are present in vivo in thepatient. The method does not involve a separate step of exposing themacrophages to the Aβ protein, in vitro, after extraction from thepatient.

In some embodiments, the level of the Aβ protein fragments detected iscompared with the level detected in a control individual who does nothave AD. In such embodiments, a comparison is made between the level andpattern of Aβ protein fragments from the patient and those of thecontrol individual. Where the level of Aβ protein fragments issignificantly below that in the control individual then this isindicative of AD in the patient. Similarly, if the type of Aβ proteinfragments present in the individual is significantly different fromthose in the control individual then this is indicative of AD in thepatient. In alternative embodiments, a standard level of Aβ proteinfragments is generated by detecting the presence of such fragments inthe macrophages in CSF in a plurality of control individuals who do nothave AD. The level and pattern of Aβ protein fragments from the patientis then compared with the standard level and a statistically significantreduction in level or difference in pattern of the presence of fragmentsis indicative of AD.

In other embodiments, a single patient is examined annually over aperiod of time (e.g. 10 years). On each occasion, the levels of Aβprotein fragments in the macrophages in a CSF sample from the patient isstudied as described above. A significant change in the level or patternof Aβ protein fragments each year, in particular a reduction in thelevel of the Aβ protein fragments year on year, is indicative of thepresence of AD.

In certain embodiments, additional AD markers in the patient are alsomeasured, at the same time as the above-described analysis is carriedout. Such additional AD markers include abnormal levels of Aβ42, Tau,Phospho-Tau or the Aβ42/Aβ40 ratio in a CSF sample obtained from thepatient or in the RNA profile of a blood or CSF sample obtained from thepatient. An abnormal level of some or all of these additional AD markersas well as an abnormal level of Aβ protein fragments in the macrophagesin the CSF of the patient is indicative of the presence of AD. Anexemplary abnormal (i.e.

pathological) level of Aβ42 is a CSF concentration of less than 550pg/ml. The concentration of Tau is age-dependent, high levels beingpathological. A pathological level of Phospho-Tau is a CSF concentrationof greater than 85 pg/ml. A pathological level of the Aβ42/Aβ40 ratio iswhere (Aβ42/Aβ40)×10 is less than 1.

In the above described embodiments, a CSF sample from the patient isobtained.

However, in alternative embodiments, a different type of sample isstudied, for example, a blood sample. Such an alternative sample may beused because macrophages circulate from the bone marrow to the CNS andtherefore macrophages in the blood of a patient may have been exposed toproteins in the CNS. It is, of course, easier to obtain a blood samplethan a CSF sample from a patient.

One embodiment of the present invention uses the following criteria asthe basis of a diagnostic test to assess Alzheimer's disease in apatient's activated macrophages/microglia: 1) Fulfillment of diseasecriteria, 2) Presence and sorting of CD16+ population of cells in CSFand blood with flow cytometry, 3) Presence/absence of Aβ peptidefragments in MS spectra after immunoprecipitation with antibodies, 4)Tailored methods at clinics. Flow cytometry and IP-MS can be replaced byother methods for sorting or distinguishing of cell subtypes and peptidefragment analysis.

Methods of evaluating fulfillment of disease criteria. The patientundergoes a thorough clinical investigation, including a study ofmedical history, physical, neurological and psychiatric examination,screening laboratory tests and MRI and PET imaging of the brain. Thediagnosis of AD is made according to recently published criteria [12].The patient undergoes a thorough physical and psychological examinationwhen enrolled in the diagnosis program at a hospital. The examinationincludes neuropsychological questionnaires for identification ofcognitive deficits, neurological examination, genetic analysis, CSFbiomarkers, imaging and metabolic profile.

Methods of evaluating presence and sorting of CD 16+ population of cellsin CSF and blood with flow cytometry. Cells are acquired on a FACSAriaCell-Sorting System and analyzed using FACSDiva software (both BectonDickinson). CSF cell populations are sorted based on their expression ofrelevant surface markers (CDs). Cells are gated according to forward-and side light-scattering properties and are positively selected for thepresence of CD45 + CD3 + CD4 + CD8 (characterization of T-cellpopulation), and CD45 + CD14 + CD16 + CD 19 (characterization ofactivated macrophages and B-cell population). In order to preserve theimmune cells intact, the cell sorting is performed at a maximum of fourhours post puncture. CD14⁺/CD16⁺ sorted cells are lysed and kept frozenat −80° C. for further analysis (protein-analysis). In addition tocollecting cells for protein analyses, the flow cytometry resultsindicate the CSF and periphery (blood) immune cell distribution for thepatient.

Method of preparation of cells for immunoprecipitation. CSF cellpopulations are sorted based on their expression of relevant surfacemarkers (CDs). Cells are gated according to forward- and sidelight-scattering properties and are positively selected for the presenceof CD45 + CD3 + CD4 + CD8 (characterization of T-cell population), andCD45 + CD 14 + CD 16 + CD 19 (characterization of activated macrophagesand B-cell population). Cell population and number of cells within eachpopulation are obtained and registered. The number of activated cells in7-10 days post stroke patients is high, suggesting circulation ofrecruited cells also to the CSF compartment of a large number of immunecells. The number of activated macrophages in AD largely equals that inthe MCI/non-AD group. The total of %-activated cells in MS is lower thatAD; which may be because the immune process in MS mainly involvesT-cells. The sorted cells are washed with 400 μl PBS and centrifuged (4°C., 750×g, 5 min). The supernatant is removed and prepared for IP-MSanalysis by adding 10 μL RIPA-buffer for cell lysis and keeping frozenat −80° C. prior to protein-analysis.

Method of immunoprecipitation. An aliquot (4 μg) of the monoclonalantibodies 6E10 (1 mg/mL, epitope 4-9), 4G8 (1 mg/mL, epitope 18-22), or11A50-B10 (0.5 mg/mL, reactive to the C-terminus) (Signet Laboratories,Inc.) is separately added to 50 μL magnetic Dynabeads (Sheep anti mouse,IgG) and incubated overnight on a rocking platform at +4° C. Theremaining unbound antibody is removed by washing twice withphosphate-buffered saline (PBS, pH 7.4). After adding 1 mL CSF to theantibody-coated beads, the incubation is continued for an additional 1 hat +4° C. The beads are pelleted for 5 min by using a magnetic particleconcentrator (Dynal MPC) and washed twice with PBS (pH 7.4) and twicewith 50 mM ammonium bicarbonate (pH 7.3). After the final wash, theextracted Aβ peptides are eluted by adding 20 μL 0.5% formic acid (FA)in water. After vortexing for 2 min in room temperature, the beads arepelleted using the magnetic particle concentrator and the supernatant iscollected. The collected supernatant is dried down in a vacuumcentrifuge and redissolved in 5 82 L 0.1% FA in 20% acetonitrile (ACN).All solvents used are of HPLC quality and all aqueous solutions are madeusing 18.2 M deionized water obtained from a Millipore purificationsystem.

Methods of evaluating presence/absence of Aβ in MS spectra afterimmunoprecipitation. IP-MS is used to isolate and determine the Aβpeptide content (Aβ signature) in the CD14⁺/CD16⁺ macrophages sorted byflow cytometry. Proteolytically processed Aβ peptides are difficult todetect using standard proteomic methods possibly because they comprise aheterogeneous set of both N- and C-terminally truncated peptides, someat low quantity. IP-MS analysis has been used previously to obtain an Aβpeptide signature successfully [43] [44]. Briefly, the Aβ peptides areisolated from lysed macrophages using anti Aβ monoclonal antibodies andmagnetic Dynabeads. Then a matrix-assisted laser desorption/ionizationtime of flight mass spectrometry (MALDI-TOF MS) analysis is performed onthe immunoprecipitated peptides and the macrophage Aβ signature iscalculated. The Absence of Aβ signal in the specimen is interpreted as apositive AD diagnosis.

Alternative Methodologies. In variants of the above-describedmethodology, the following techniques are used.

1. Instead of using flow cytometry to sort cells, activatedmacrophages/microglia cells are withdrawn using magnetic extraction,flotation techniques, or other antibody or affinity-based extractiontechniques e.g. chromatography, gradient centrifugation. Alternativelythe cells are studied using immunohistochemistry

2. Immunoprecipitation using other antibodies specific for thepeptide/protein of interest.

3. Instead of using mass spectrometry, another technique forquantitative or semiquantitative peptide/protein analysis is employedsuch as: HPLC-fluorescence or -UV, luminescence, streptavidin/biotinsystems, immunohistochemistry.

Alternative Conditions. In alternative embodiments a differentpathological condition characterized by the presence of fragments of amarker protein in the brains of patients is studied. In each case it isnecessary to identify the condition to be studied and the correspondingprotein that characterizes the condition. Exemplary conditions include:Parkinson's Disease in which ubiquitin is the characterizing protein;Multiple Sclerosis where myelin basic protein characterizes thecondition; FrontoTemporal Dementia and Amyotrophic Lateral Sclerosiswhich are characterized by the tau protein; and Parkinson's Disease,Lewy body dementia and AD which are characterized by the alpha-synucleinprotein. In each case, the method of detection or monitoring is carriedout as is described above in relation to AD except that the antibodiesused to immunoprecipitate the peptides from the macrophages aresubstituted with antibodies that are capable of binding fragments of thecharacterizing protein of the condition. Furthermore, in the case ofMultiple Sclerosis, abnormally high levels of the ubiquitin markerprotein are indicative of the presence of the condition.

In some embodiments, multiple such conditions are tested forsimultaneously by immunoprecipitating cell lysates with multiple sets ofantibodies, each set of antibodies being specific for fragments ofdifferent characterizing proteins.

In some embodiments of the invention, a diagnostic kit is provided inorder to enable the detection of a pathological condition of theinvention (that is to say a condition characterized by the presence offragments of a marker protein in the brain of a patient suffering fromthe condition). The kit is suitable for use in ordinary clinicallaboratories since it is based on an ELISA/immuno-PCR technique and sodoes not require the use of MALDI-TOF or IP-MS techniques as describedin some previous embodiments. The kit comprises a panel of targetspecific antibodies which are specific for a first epitope of the markerprotein. Thus, for example, where the pathological condition to bedetected is Alzheimer's disease, the marker protein is the Abeta 42protein. The kit also comprises a supply of magnetic beads which displaymacrophage specific antibodies (for example, antibodies specific for theCD 14 and CD 16 cell markers); a cell lysing agent such as RadioImmunoPrecipitation Assay (RIPA) Buffer containing 25 mM Tris-HCl pH 7.6, 150mM NaCl, 1% NP-40, 1% Sodium deoxycholate and 0.1% SDS (PierceBiotechnology); and a secondary antibody which is specific for a secondepitope of the marker protein. The secondary antibody is conjugated to adouble-stranded DNA marker molecule.

The kit will now be described in use. A sample, such as a peripheralblood or CSF sample, is obtained from a patient and macrophage cells areisolated from the sample by mixing with the magnetic beads provided inthe kit. The macrophage specific antibodies displayed by the magneticbeads bind the macrophages in the patient sample and the macrophages andthe magnetic beads are then removed from the sample by magnetic means.The macrophage cells are then released from the macrophage specificantibodies by adjusting the pH of the solution and the macrophage cellsare lysed with the lysing agent in order to release the cell contentswhich includes the marker protein. Also provided in the diagnostic kitis a solid support on which are immobilized a plurality of targetantibodies which are specific for the marker protein. The contents ofthe lysed macrophage cell are then contacted with the solid support suchthat the first epitope of the marker protein binds to the targetantibody.

The solid support is contacted with a secondary antibody which isconjugated to a double-stranded DNA marker molecule. The secondaryantibody is specific for the second epitope of the marker protein suchthat the secondary antibody is immobilized on the solid support wherethe marker protein is present. Unbound proteins and unbound secondaryantibody are then washed out and removed.

The washed solid support is then subjected to real time PCR which meltsthe double stranded DNA marker molecule and amplifies the copy number inorder to identify the number of copies of the DNA marker molecule. Thenumber of copies of the DNA marker molecule after a predetermined numberof cycles of PCR amplification is indicative of the starting number ofDNA molecules. Furthermore, there is a one-to-one relationship betweenthe starting number of DNA molecules and the number of bound markerproteins. Therefore, this immuno-PCR technique provides an accurateindication of the number of marker protein molecules in the patientsample.

Accordingly, such diagnostic kits allow a simple immunological method tobe used in standard clinical laboratories which are available in allhospitals, private clinics and commercial laboratories in order toanalyze patient samples in accordance with the present invention. Theuse of the kit of the invention does not require the use of expensive oradvance laboratory instruments and detection using an immuno-PCRtechnique ensures high sensitivity.

In some variants of the above-described diagnostic kits, a plurality ofpanels of antibodies are provided in the kit. For example, in onevariant, the kit comprises first target antibodies that are specific fora first epitope (e.g., SEQ ID Nos:1-5) of the Aβ protein and secondtarget antibodies which are specific for a second epitope of the Aβprotein (e.g., SEQ ID Nos:1-5). In still further embodiments, aplurality of panels of antibodies are provided in the kit and theantibodies are specific for marker proteins corresponding to more thanone pathological condition. For example, in one particular variant, apanel of antibodies is provided which is specific for the Abeta protein(the marker protein for Alzheimer's disease) and a panel of antibodiesis provided specific for Multiple Sclerosis (where myelin basic proteinis the marker protein). In these variants, it is preferred thatdifferent panels of secondary antibodies, each specific for a respectivemarker protein and each conjugated to a different DNA marker molecule,are provided such that the signal for the detection of each markerprotein is distinguishable.

In the above described embodiments of the diagnostic kit, the detectablelabel is a DNA marker molecule. However, in other embodiments, adifferent detectable label is used. For example, the detectable labelmay be a fluorophore, a latex microbead or a gold particle. Suchalternative detectable labels are useful when the kit is provided onlyto provide a qualitative result rather than a quantitative result.

In some alternative embodiments of the kit, a lysing agent, as such, isnot provided. Instead, cells are lysed mechanically, e.g. bycentrifugation, prior to isolation of the macrophages.

It is also to be appreciated that the diagnostic kits of the presentinvention are not limited to kits comprising antibodies. In alternativeembodiments, the antibodies of the kit are replaced with other bindingreagents such as antigen binding fragments (e.g. F(ab′)₂ fragments orFab fragments) or a polynucleotide sequence. Typically such otherbinding reagents have binding affinities for their target comparable tothat of antibodies such as having a binding affinity of less than 100 nmin an aqueous buffered solution at between pH 4 and 8.

EXAMPLES

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

Example 1 Identification of Amyloid Beta (Aβ) Peptides in IsolatedMonocytes/Macrophages

Peripheral blood monocytes/macrophages from a healthy volunteer wereisolated and incubated with addition of synthetic Aβ₄₂ in the growthmedium overnight to facilitate uptake and phagocytosis of Aβ₄₂. Acontrol sample without addition of Aβ₄₂ was also included. Samplecollection and monocyte/macrophage isolation was as described in in PCTApp. No. PCT/EP09/001210, herein incorporated by reference in itsentirety.

After cells were harvested, cellular organelles (ER, mitochondria,endosomes, lysosomes etc) were extracted and subsequently separated onan Iodixanol gradient by ultracentrifugation. Polypeptides present inthe organelle fraction corresponding to the lysosomes were analyzedusing mass spectrometric analysis with database searches based upon theAβ42 amino acid sequence.

Several Aβ₄₂ fragments were identified and found to be specific for theAβ₄₂-phagocytosis condition. In addition, two shorter peptides wereunequivocally identified and still sufficiently long to highly likelypresent antigenic epitopes, the stretches GSNKGAI (25-32 of the Aβ42molecule, corresponding to the sequence 621-627 of the APP, and FAEDVG(20-25 of the Aβ42 molecule, corresponding to 616-621 in APP) bothpartially in the transmembrane region (FIG. 4).

Five new Aβ₁₋₄₂fragments were identified and these highly likely presentantigenic epitopes. These fragments are depicted as SEQ ID NOs: 1-5 inFIG. 2 and have the following amino acid sequences:

SEQ ID NO: 1: FAEDVG (20-25 of the Aβ₁₋₄₂molecule, corresponding to616-621 in APP) SEQ ID NO: 2: AEDVGSNKGAI(21-31 of the Aβ₁₋₄₂molecule, corresponding tothe sequence 617-627 of the APP) SEQ ID NO: 3: GSNKGAIIGLM(25-35 of the Aβ₁₋₄₂molecule, corresponding to 621-631 in APP)SEQ ID NO: 4: GSNKGAI (25-32 of the Aβ₁₋₄₂molecule, corresponding tothe sequence 621-627 in APP) SEQ ID NO: 5: VGSNKGAIGL(24-34 of the Aβ₁₋₄₂molecule, corresponding tothe stretch 620-630 in APP)

All fragments are located within the intramembrane region of the Aβ₁₋₄₂molecule. While the present invention is not limited to any particularmechanism, and an understanding of the mechanism is not necessary topractice the present invention, it is contemplated that they are likelyto be generated by Aβ-cleaving enzymes such as insulin degrading enzyme(IDE) and neprilysin (Malito et al. (2008) Cell Mol Life Sci,65:2574-2585; herein incorporated by reference in its entirety

Example 2 Synthesis of Antigens

Based on the experiment described in Example 1, the following peptideswere selected for further study and produced by Eurogentec, (MedProbeNorway):

Antigen 1 (SEQ ID NO: 2): H₂N-AED VGS NKG AI-CONH₂Antigen 2 (SEQ ID NO: 3): H₂N-GSN KGA IIG LM-CONH₂Antigen 3: (SEQ ID NO: 5): H₂N-VGS NKG AIG L-CONH₂

Example 3 Immunization of Mice in Order to Obtain Response Towards theSynthetic Peptides. Establishment of Clones Producing MonoclonalAntibodies Against Alzheimer-Specific Aβ₁₋₄₂ Metabolites from within theLysosomes of Monocytes

Animals: 6 BALB/c female mice, 10 NMRI mice

The BALB/c mice were immunized 27/12-10 with the regime outlined inTable 1. A second immunisation (boost) was performed 31/1-11, the sameregime applied without adjuvants and the peptide was injected IP and IV.Blood was drawn from the mice and analysed at Diatec Monoclonals AS forpresence of specific antibodies. Macrophages from the NMRI mice servedas feeder cells for antibody production in vitro. Screening of bloodsamples from the mice nr 1-6 against the mix of antigens with or withoutKLH resulted in positive hits from mouse nr 1, 4 and 5 and negative hitsfrom mouse nr 6. Blood from mouse nr 2 was negative while blood frommouse nr 3 was strongly positive for the peptide mix including KLH,indicating an immune response towards KLH instead of the peptides.Spleens from the three positive mice+ the control mouse in Week 7 2011were extracted, thus concluding the animal work without a thirdimmunization boost.

TABLE 1 Mouse nr Peptide Adjuvant 1 Ag1 200 μL IP Freunds adjuvantcomplete 2 Ag2 200 μL IP 200 μL IP 3 Ag1 66.7 μL IP (1:1 emulsion withpeptide) 4 Ag2 66.7 μL IP 5 Ag3 66.7 μL IP 6 PBS 200 μL IP

The spleens of mice that included antibodies against the peptides weredisintegrated and cells were incubated with feeder cells from NMRI-micefor clonal expansion. After ELISA and a 2^(nd)recloning of selectedclones showing good specificity against the antigen clone 6D3/1/15(mouse 5), 2A3/1/13 (mouse 1) and 3B7/1/16 (mouse 5) gave good results.All three clones gave specific response against antigen 1 and wereincluded in a cellbank and master cell bank (MCB).A test production fromone (from the MCB) ampoule from each clone are performed. Concentration:1 mg/mL, 0.09% azide. 3B7/2/6 (6.7 mL), 2A3/2/15 (12.2 mL), 6D3/2/22 (28mL). Delivered 29.06.2011. Isotype Mouse IgG.

Example 4 Measurement of Antigen Presence in Biological Sample UsingTHP-1-Acute Monocytic Leukemia Cells (American Type Culture Collection(ATCC)

FIG. 6 provides a schematic depiction of an Immune-PCR assay using theantibodies described in Example 3. The capture antibody is one of thethree antibodies developed via immunization. The two other antibodieswill be directly conjugated with a specific DNA tag that is amplifiedusing real-time PCR.

For the assay, THP-1 cells are cultivated in 2 bottles with RPMI mediumcontaining 10% FBS and 1% antibiotic. The cells are differentiated usinga final concentration of 100 nM TPA for 17 h. To one bottle is addedsynthetic Aβ₁₋₄₂ to a final concentration of 2.5 ng/μL, the other keptuntreated. The cells are incubated over night at 37° C. in a humidifiedatmosphere with 5% CO₂.The cells are then washed and trypsinated(Trypsin-Versene (EDTA) from Lonza) in order to release the attachedcells from the bottom surface.

Flowcytometry of THP-1 cells. The cells are counted and four tubescontaining 100 000 cells each are subjected to fixation andpermeabilization using the IntraPrep kit from Beckman Coulter, Inc, USAto allow for staining of intracellular targets. Two tubes (one tube thathas received Aβ and one untreated) are stained with two commerciallyavailable monoclonal antibodies against Aβ₁₋₄₂ (6E10 and 4G8 originatingfrom mice) and two tubes (one tube that has received Aβ and oneuntreated) are stained with appropriate isotype control antibodies(Mouse IgG1 and IgG2b). All these antibodies are obtained from NordicBioSite AS, Norway. The secondary antibody AlexaFluor488-Goat anti-Mouse(Invitrogen Dynal AS, Norway) is used to detect both the An-specificantibodies and the isotype controls. The cells are kept in IOTest3Fixative solution (Beckman Coulter, INC, USA) at 4° C. over night beforeflow cytometric analysis on a FACS Cantoll from BD. The cells arefinally acquired and gated based on scatter and fluorescent properties.Monocytes/macrophages positive for AlexaFluor488, i.e. with higherfluorescent signal than isotype controls, are considered Aβ-positive.Analysis is performed using FACS Diva software (BD).

Preparation of samples for immuno-PCR. The remaining cells (approx. 319000 cells receiving Aβ₁₋₄₂ and approx. 560 000 cells untreated) arecentrifuged at 1000 g for 10 min. The supernatant are removed and 404lysing agent, M-PER® (Mammalian protein extraction reagent from Pierce))added 1% protease inhibitor cocktail (Sigma Life Sciences) are added tothe cell pellet.

The lysates are incubated in a mixer for 30 minutes at room temperature.After incubation the samples are kept frozen until analysis.

The detection antibody 3B7/2/6 is biotinylated in house (Lightning-LinkBiotin Conjugation Kit Type A (Innova Biosciences)) in order to bind toanti-biotin detection conjugate.

The development of immuno-PCR is performed using the Imperacer® assaydevelopment kit (Chimera Biotec, Germany).

Assay pre-preparation—Immobilization of capture antibody. Antibody6D3/2/22 is diluted in coating buffer (final concentration 10 μg/ml). 30μL of diluted antibody are added into each well. The wells are sealedwith foil and incubated for at least 16 hours at 4° C. The wells arewashed and chimera direct block (240 μL/well) is added for 30-60 secondsat room temperature with orbital shaking. 30 μL of four dilutions usingthe synthesized antigen 1 are added to each well (0 ng/mL, 0.1 ng/mL, 1ng/mL, and 10 ng/mL) as standards. 30 μL of lysate from Aβ treated anduntreated cells is added to each well. The samples are incubated for 30minutes at room temperature during orbital shaking.

After incubation of the analyte, 30 μL of biotinylated primary detectionantibody (antibody 3B7/2/6, final concentration 1 μg/ml) is added to thesample in order to be coupled to the immobilized analyte. The samplesare incubated for 30 minutes at room temperature and orbital shaking.

After incubation with primary detection antibody, 30μL/well of dilutedImperacer® Conjugate CHI-Biotin (1:3000) is added to the samples. Thesamples are incubated for 30 minutes at room temperature with orbitalshaking.

The samples are washed and 35 μL of PCR mastermix is added. The wellsare then incubated at 95° C. for 5 minutes. After incubation 30 μl ofthe samples are transferred to the reaction plate (Micro Amp FastOptical 96-well Reaction Plate (Applied Biosystems)). In addition tostandards and samples a positive and negative PCR control are includedin the reaction plate.

Real-time PCR. The PCR is carried out using 7900HT Fast Real-Time PCRsystem (Applied Biosystems). FAM is set up as a fluorophor (emission at518 nm).

TABLE 2 Real-time PCR program: Time Temperature Repeats  4 minutes 95°C.  1x 12 seconds 95° C. 50x 30 seconds 50° C. 30 seconds 72° C.The results are provided in FIG. 7, FIG. 8 and Table 3.

TABLE 3 Sample Ct-value 0 ng/mL 33.497 0.1 ng/mL 33.216 1 ng/mL 30.34710 ng/mL 26.734 Cell lysate from THP-1 cells receiving Aβ 29.65 Celllysate from THP-1 untreated 30.332

The Ct value represents the first PCR cycle at which the reporter signalexceeds the signal of a given uniform “Threshold”.

Conclusion.

The Ct value for the sample receiving additional Aβ is lower than theuntreated sample. The concentration for this sample is 4, 91 ng/mL whenusing the equation for the standard curve. The concentration for theuntreated sample is 3, 79 ng/mL. This indicates a higher presence ofantigen 1 in cell lysate receiving Aβ and shows that it is possible tomeasure degradation products of Aβ in biological samples with thismethod.

Example 5 Measurement of Antigen Presence in Monocytes/Macrophages fromPatient with AD (F, Age 62) and Healthy Control (Spouse)(M, Age 60)

Heparinized blood (8 ml) is drawn by venipuncture. From the bloodsample, peripheral blood mononuclear cells (PBMC's) are isolated withHistopaque®1077 from Sigma-Aldrich Corporation, USA, i.e. thegranulocytes and the red blood cells are excluded from the followingsteps. The monocytes/macrophages are isolated from the other cells usingflow cytometry. The cells are analyzed on a FACS Aria from BD. The cellsare acquired and gated based on forward scatter (size) and side scatter(granularity) properties. The monocyte/macrophage population is sortedinto dedicated tubes (Patient and Control).

Flowcytometry of Monocytes/Macrophages from Patient and Control

Intracellular staining of macrophages/monocytes from patient and controlusing commercially available monoclonal antibodies against Aβ₁₋₄₂. Allcells subjected to fixation and permeabilization using the IntraPrep kitfrom Beckman Coulter, Inc, USA to allow for staining of intracellulartargets. One tube (100 000 PBMC) from both patient and control isstained with two commercially available monoclonal antibodies againstAβ42 (6E10 and 4G8 originating from mice) and one tube from both patientand control are stained with appropriate isotype control antibodies(Mouse IgG1 and IgG2b). All these antibodies are obtained from NordicBioSite AS, Norway. The secondary antibody AlexaFluor488-Goat anti-Mouse(Invitrogen Dynal AS, Norway) is used to detect both the Aβ-specificantibodies and the isotype controls. The cells are kept in IOTest3Fixative solution (Beckman Coulter, INC, USA) at 4° C. for up to 5 daysbefore flow cytometric analysis on a FACS Cantoll from BD.Monocytes/macrophages positive for AlexaFluor488, i.e. with higherfluorescent signal than isotype controls, are considered Aβ-positive.Analysis is performed using FACS Diva software (BD).

Preparation of Cells for Immuno-PCR

Monocytes/macrophages (440 000 cells) are centrifuged at 1200 g for 10min. The supernatant are removed and 50 μL lysing agent, M-PER®(Mammalian protein extraction reagent from Pierce) added 1% proteaseinhibitor cocktail (Sigma Life Sciences) are added to the cell pellet.

The lysates are incubated in a mixer for 30 minutes at room temperature.After incubation the samples are kept frozen until analysis.

Immuno-PCR.

Immuno-PCR is conducted as described above. 30 μL of four dilutionsusing the synthesized antigen 1 are added to each well (0 ng/mL, 0.1ng/mL, 0.5 ng/mL, and 1.0 ng/mL) as standards.

Results example 5

Results from the flowcytometry analysis are provided in FIG. 10(histograms 10 a and 10 b).

Real-time PCR

Results are provided in table 4 and FIG. 11.

TABLE 4 Sample Ct-value 0 ng/mL 38.005 0.1 ng/mL 36.419 0.5 ng/mL 34.7081 ng/mL 33.736 Patient 36.415 Control 35.71The Ct value represents the first PCR cycle at which the reporter signalexceeds the signal of a given uniform “Threshold”.Conclusion: The Ct value for the control is lower than for the patient.The concentration for the control is 0, 40 ng/mL when using the equationfor the standard curve. The concentration for the patient is 0, 22 ng/m,showing a higher amount of antigen 1 in cells from control compared topatient. Although both patient and control seems to have close to equalamount of intracellular Aβ₁₋₄₂ as shown with flowcytometry, thisreal-time PCR result indicates that this method can be used to measurespecific degradation products in biological samples differentiatingpatient from controls.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled indiagnostic assays, the medical arts, or related fields are intended tobe within the scope of the following claims.

1-20. (canceled)
 21. A method for detecting peptides in a sample, saidmethod comprising: a) obtaining a body fluid sample from a patient; andb) detecting the presence of a peptide consisting of an amino acidsequence corresponding to SEQ ID NO:5.
 22. The method of claim 21,wherein the body fluid sample comprises a cell type selected from thegroup consisting of leucocytes, monocytes, peripheral blood macrophages,and activated macrophages from peripheral blood.
 23. The method of claim22, wherein said cell type is a monocyte.
 24. The method of claim 23,wherein said monocyte is from peripheral blood.
 25. The method of claim21, wherein said body fluid sample is serum.
 26. The method of claim 21,wherein said body fluid sample is plasma.
 27. The method of claim 21,wherein said body fluid sample is cerebrospinal fluid.
 28. The method ofclaim 21, wherein said peptide has both n-terminal and c-terminaltruncations as compared to Aβ₄₂.
 29. The method of claim 21, whereinsaid detecting is performed in conjunction with monitoring efficacy of atreatment.